Scientific Resources

Azidothymidine (AZT) is a synthetic,chain-terminating nucleoside analog used to treat HIV-1 infection. While AZT is not actively transported across the blood brain barrier,it does accumulate at high levels in cerebrospinal fluid,and subsequently diffuses into the overlying parenchyma. Due to the close anatomical proximity of the neurogenic niches to the ventricular system,we hypothesize that diffusion from CSF exposes neural stem/progenitor cells and their progeny to biologically relevant levels of AZT sufficient to perturb normal cell functions. We employed in vitro and in vivo models of mouse neurogenesis in order to assess the effects of AZT on developing and adult neurogenesis. Using in vitro assays we show that AZT reduces the population expansion potential of neural stem/progenitor cells by inducing senescence. Additionally,in a model of in vitro neurogenesis AZT severely attenuates neuroblast production. These effects are mirrored in vivo by clinically-relevant animal models. We show that in utero AZT exposure perturbs both population expansion and neurogenesis among neural stem/progenitor cells. Additionally,a short-term AZT regimen in adult mice suppresses subependymal zone neurogenesis. These data reveal novel negative effects of AZT on neural stem cell biology. Given that the sequelae of HIV infection often include neurologic deficits-subsumed under AIDS Dementia Complex (Brew,1999)-it is important to determine to what extent AZT negatively affects neurological function in ways that contribute to,or exacerbate,ADC in order to avoid attributing iatrogenic drug effects to the underlying disease process,and thereby skewing the risk/benefit analysis of AZT therapy. View Publication

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique,we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition,we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells,but not in transit-amplifying cells,and directly impacts on neurogenesis. Finally,we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFβ regenerative therapies based on stimulating endogenous neural stem cells. View Publication

Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals,they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However,little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ),we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months,the proliferative status of activated NSCs was already altered by 6 months,with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures,as well as a modulation of the TGFβ signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications. View Publication

Quiescent neural stem cells (NSCs) are considered the reservoir for adult neurogenesis,generating new neurons throughout life. Until now,their isolation has not been reported,which has hampered studies of their regulatory mechanisms. We sorted by FACS quiescent NSCs and their progeny from the subventricular zone (SVZ) of adult mice according to the expression of the NSC marker LeX/CD15,the EGF receptor (EGFR) and the CD24 in combination with the vital DNA marker Hoechst 33342. Characterization of sorted cells showed that the LeX(bright)/EGFR-negative population was enriched in quiescent cells having an NSC phenotype. In contrast to proliferating NSCs and progenitors,the LeX(bright)/EGFR-negative cells,i.e. quiescent NSCs,resisted to a moderate dose of gamma-radiation (4Gy),entered the cell cycle two days after irradiation prior to EGFR acquisition and ultimately repopulated the SVZ. We further show that the GABAAR signaling regulates their cell cycle entry by using specific GABAAR agonists/antagonists and that the radiation-induced depletion of neuroblasts,the major GABA source,provoked their proliferation in the irradiated SVZ. Our study demonstrates that quiescent NSCs are specifically enriched in the LeX(bright)/EGFR-negative population,and identifies the GABAAR signaling as a regulator of the SVZ niche size by modulating the quiescence of NSCs. View Publication

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36),an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly,TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand,inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. View Publication

Adeno-associated virus (AAV) vectors expressing tumoricidal genes injected directly into brain tumors have shown some promise,however,invasive tumor cells are relatively unaffected. Systemic injection of AAV9 vectors provides widespread delivery to the brain and potentially the tumor/microenvironment. Here we assessed AAV9 for potential glioblastoma therapy using two different promoters driving the expression of the secreted anti-cancer agent sTRAIL as a transgene model; the ubiquitously active chicken β-actin (CBA) promoter and the neuron-specific enolase (NSE) promoter to restrict expression in brain. Intravenous injection of AAV9 vectors encoding a bioluminescent reporter showed similar distribution patterns,although the NSE promoter yielded 100-fold lower expression in the abdomen (liver),with the brain-to-liver expression ratio remaining the same. The main cell types targeted by the CBA promoter were astrocytes,neurons and endothelial cells,while expression by NSE promoter mostly occurred in neurons. Intravenous administration of either AAV9-CBA-sTRAIL or AAV9-NSE-sTRAIL vectors to mice bearing intracranial patient-derived glioblastoma xenografts led to a slower tumor growth and significantly increased survival,with the CBA promoter having higher efficacy. To our knowledge,this is the first report showing the potential of systemic injection of AAV9 vector encoding a therapeutic gene for the treatment of brain tumors. View Publication

Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here,we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system,we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly,the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH,hormones used in therapeutic treatment in humans. Finally,we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus,we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons. View Publication

BACKGROUND In glioblastoma (GBM),Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance. METHODS Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2),a stabilized form of PGE2,to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1,a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting,quantitative real-time (qRT)-PCR,and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM. RESULTS In GBM cells,dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads,in turn,to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage,which are dependent on Id1. CONCLUSIONS In GBM,Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively,these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients. View Publication

Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are good candidates for cell therapy due to the accessibility of fat tissue and the abundance of AT-MSCs therein. Neurospheres are free-floating spherical condensations of cells with neural stem/progenitor cell (NSPC) characteristics that can be derived from AT-MSCs. The aims of this study were to examine the influence of oxygen (O2) tension on generation of neurospheres from canine AT-MSCs (AT-cMSCs) and to develop a hypoxic cell culture system to enhance the survival and therapeutic benefit of generated neurospheres. AT-cMSCs were cultured under varying oxygen tensions (1%,5% and 21%) in a neurosphere culture system. Neurosphere number and area were evaluated and NSPC markers were quantified using real-time quantitative PCR (qPCR). Effects of oxygen on neurosphere expression of hypoxia inducible factor 1,α subunit (HIF1A) and its target genes,erythropoietin receptor (EPOR),chemokine (C-X-C motif) receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF),were quantified by qPCR. Neural differentiation potential was evaluated in 21% O2 by cell morphology and qPCR. Neurospheres were successfully generated from AT-cMSCs at all O2 tensions. Expression of nestin mRNA (NES) was significantly increased after neurosphere culture and was significantly higher in 1% O2 compared to 5% and 21% O2. Neurospheres cultured in 1% O2 had significantly increased levels of VEGF and EPOR. There was a significant increase in CXCR4 expression in neurospheres generated at all O2 tensions. Neurosphere culture under hypoxia had no negative effect on subsequent neural differentiation. This study suggests that generation of neurospheres under hypoxia could be beneficial when considering these cells for neurological cell therapies. View Publication

The cell surface marker CD133 has been proposed as a brain tumor stem cell marker. However,there have been substantial controversies regarding the necessity and role of CD133 in tumorigenesis. This study aimed to characterize CD133(+) cells in brain tumors. Human brain tumor specimens and whole blood were collected from the same patients (N=12). We carried out dual FACS staining for CD133/CD34 and functional tumorigenesis and angiogenesis analyses of CD133(+) cells from different origins. We also investigated the in vivo tumorigenic potential and histological characteristics of four distinct groups on the basis of expression of CD133/CD34 markers (CD133(+),CD133(+)/CD34(+),CD133(+)/CD34(-),and CD133(-)). CD133(+) brain tumor cells coexpressed significantly higher positivity for CD34 (70.7±5.2% in CD133(+) vs. 12.3±4.2% in CD133(-) cells,P<0.001). CD133(+) brain tumor cells formed neurosphere-like spheroids and differentiated into multiple nervous system lineages unlike CD133(+) blood cells. They showed biological characteristics of endothelial cells,including vWF expression,LDL uptake and tube formation in vitro,unlike CD133(-) brain tumors cells. Pathologic analysis of brains implanted with CD133(+) cells showed large,markedly hypervascular tumors with well-demarcated boundary. CD133(+)/CD34(-) cells produced smaller but highly infiltrative tumors. Notably,pure angiogenic cell fractions (CD133(+)/CD34(+)) and CD133(-) tumor cells did not generate tumors in vivo. Our data suggest the presence of a distinct subpopulation of CD133(+) cells isolated from human brain tumors,with characteristics of endothelial progenitor cells (EPCs). View Publication

Neurogenesis in the adult brain is important for memory and learning,and the alterations in neural stem cells (NSCs) may be an important part of Alzheimer's disease pathogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway has been suggested to play an important role in neuronal cell survival and is highly involved in adult neurogenesis. Recently,coenzyme Q10 (CoQ10) was found to affect the PI3K pathway. We investigated whether CoQ10 could restore amyloid β (Aβ)25-35 oligomer-inhibited proliferation of NSCs by focusing on the PI3K pathway. To evaluate the effects of CoQ10 on Aβ25-35 oligomer-inhibited proliferation of NSCs,NSCs were treated with several concentrations of CoQ10 and/or Aβ25-35 oligomers. BrdU labeling,Colony Formation Assays,and immunoreactivity of Ki-67,a marker of proliferative activity,showed that NSC proliferation decreased with Aβ25-35 oligomer treatment,but combined treatment with CoQ10 restored it. Western blotting showed that CoQ10 treatment increased the expression levels of p85α PI3K,phosphorylated Akt (Ser473),phosphorylated glycogen synthase kinase-3β (Ser9),and heat shock transcription factor,which are proteins related to the PI3K pathway in Aβ25-35 oligomers-treated NSCs. To confirm a direct role for the PI3K pathway in CoQ10-induced restoration of proliferation of NSCs inhibited by Aβ25-35 oligomers,NSCs were pretreated with a PI3K inhibitor,LY294002; the effects of CoQ10 on the proliferation of NSCs inhibited by Aβ25-35 oligomers were almost completely blocked. Together,these results suggest that CoQ10 restores Aβ25-35 oligomer-inhibited proliferation of NSCs by activating the PI3K pathway. View Publication

Premature proliferative arrest in benign or early-stage tumors induced by oncoproteins,chromosomal instability,or DNA damage is associated with p53/p21 activation,culminating in either senescence or apoptosis,depending on cell context. Growth hormone (GH) elicits direct peripheral metabolic actions as well as growth effects mediated by insulin-like growth factor 1 (IGF1). Locally produced peripheral tissue GH,in contrast to circulating pituitary-derived endocrine GH,has been proposed to be both proapoptotic and prooncogenic. Pituitary adenomas expressing and secreting GH are invariably benign and exhibit DNA damage and a senescent phenotype. We therefore tested effects of nutlin-induced p53-mediated senescence in rat and human pituitary cells. We show that DNA damage senescence induced by nutlin triggers the p53/p21 senescent pathway,with subsequent marked induction of intracellular pituitary GH in vitro. In contrast,GH is not induced in cells devoid of p53. Furthermore we show that p53 binds specific GH promoter motifs and enhances GH transcription and secretion in senescent pituitary adenoma cells and also in nonpituitary (human breast and colon) cells. In vivo,treatment with nutlin results in up-regulation of both p53 and GH in the pituitary gland,as well as increased GH expression in nonpituitary tissues (lung and liver). Intracrine GH acts in pituitary cells as an apoptosis switch for p53-mediated senescence,likely protecting the pituitary adenoma from progression to malignancy. Unlike in the pituitary,in nonpituitary cells GH exerts antiapoptotic properties. Thus,the results show that GH is a direct p53 transcriptional target and fulfills criteria as a p53 target gene. Induced GH is a readily measurable cell marker for p53-mediated cellular senescence. View Publication